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George D. Bittner - One of the best experts on this subject based on the ideXlab platform.
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phosphorylation of neurofilament proteins in isolated goldfish mauthner Axoplasm
Journal of Neurochemistry, 2002Co-Authors: Tim D Raabe, George D. BittnerAbstract:Abstract: The six neurofilament proteins (NFPs) in the goldfish Mauthner axon (M-axon) have molecular sizes of 235, 145, 123, 105, 80, and 60 kDa. To determine if NFPs in the M-axon are phosphorylated, isolated Mauthner Axoplasm (M-Axoplasm) and a neurofilament-enriched extract (NFE) prepared from M-Axoplasm were incubated with 32P, which resulted in the radiolabeling of NFPs as determined by their detection on autoradiograms. Kinase inhibitors directed against cyclic AMP-dependent kinases (PKAs) or cofactor-independent kinases significantly reduced the in vitro phosphorylation of NFPs in NFE, whereas inhibitors directed against protein kinase C did not significantly reduce the in vitro phosphorylation of NFPs in NFE. Experiments using two kinase inhibitors directed against different kinases significantly reduced the in vitro phosphorylation of NFPs in NFE to a greater extent than the reduction produced using any single kinase inhibitor. These data suggest that NFPs in the M-axon are phosphorylated and that the in vitro (and perhaps the in vivo) phosphorylation of NFPs is mediated by PKA and/or cofactor-independent kinases that copurify with NFPs.
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heat shock proteins in Axoplasm high constitutive levels and transfer of inducible isoforms from glia
The Journal of Comparative Neurology, 1998Co-Authors: Rebecca A. Sheller, Mark E. Smyers, Robert M Grossfeld, Martis L Ballinger, George D. BittnerAbstract:To characterize heat-shock proteins (HSPs) of the 70-kDa family in the crayfish medial giant axon (MGA), we analyzed Axoplasmic proteins separately from proteins of the glial sheath. Several different molecular weight isoforms of constitutive HSP 70s that were detected on immunoblots were approximately 1-3% of the total protein in the Axoplasm of MGAs. To investigate inducible HSPs, MGAs were heat shocked in vitro or in vivo, then the axon was bathed in radiolabeled amino acid for 4 hours. After either heat-shock treatment, protein synthesis in the glial sheath was decreased compared with that of control axons, and newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa appeared in both the Axoplasm and the sheath. Because these radiolabeled proteins were present in MGAs only after heat-shock treatments, we interpreted the newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa to be inducible HSPs. Furthermore, the 72-kDa radiolabeled band in heat-shocked Axoplasm and glial sheath samples comigrated with a band possessing HSP 70 immunoreactivity. The amount of heat-induced proteins in Axoplasm samples was greater after a 2-hour heat shock than after a 1-hour heat shock. These data indicate that MGA Axoplasm contains relatively high levels of constitutive HSP 70s and that, after heat shock, MGA Axoplasm obtains inducible HSPs of 72 kDa, 84 kDa, and 87 kDa from the glial sheath. These constitutive and inducible HSPs may help MGAs maintain essential structures and functions following acute heat shock.
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medium weight neurofilament mrna in goldfish mauthner Axoplasm
Neuroscience Letters, 1996Co-Authors: Orion D Weiner, Aaron M Zorn, Paul A Krieg, George D. BittnerAbstract:Although axons are generally considered to lack the ability to synthesize proteins, the Mauthner axon (M-axon) of the goldfish has been reported to contain some of the basic components of the translational machinery, such as transfer RNA (tRNA), ribosomal RNA (rRNA), and ribosomes. To determine if the M-axon also contains mRNA, we isolated samples of M-Axoplasm free of glial contamination as demonstrated by the absence of glial-specific mRNA and protein. Reverse transcription-polymerase chain reaction (RT-PCR) of M-Axoplasmic cDNA in the presence of primers for the goldfish medium-weight neurofilament (NF-M) gene produced a single product of the expected length for RT-PCR amplification of goldfish NF-M mRNA. This mRNA might direct protein synthesis of NF-M within the M-Axoplasm.
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Glia-to-axon communication: enrichment of glial proteins transferred to the squid giant axon.
Journal of neuroscience research, 1995Co-Authors: Rebecca A. Sheller, Michael Tytell, Mark E. Smyers, George D. BittnerAbstract:The transfer of newly synthesized proteins from the glial sheath into the axon is a well-documented process for the squid giant axon. In this study, we used a novel approach to separate the transferred glial proteins (TGPs) from the endogenous Axoplasmic proteins of the squid giant axon. Axoplasm, containing radiolabelled TGPs, was extruded as a cylinder and immersed in an intracellular buffer. After 1–30 min, the TGPs were enriched in the intracellular buffer, because they were eluted from the Axoplasm into the intracellular buffer much faster than the endogenous Axoplasmic proteins. Most of the TGPs enriched in the intracellular buffer did not pellet when centrifuged at 24,000 g for 20 min and were susceptible to protease digestion without the addition of Triton X-100. Additionally, transmission electron microscopic autoradiography of intact axons, containing radiolabelled TGPs, suggested that most TGPs were not associated with vesicular organelles within the axon. We conclude that most of the TGPs are not contained within vesicles in the Axoplasm of the squid giant axon, as would be expected if the mechanism of glia-to-axon transfer were conventional exocytosisendocytosis or microphagocytosis. © 1995 Wiley-Liss, Inc.
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long term survival of severed crayfish giant axons is not associated with an incorporation of glial nuclei into Axoplasm
Neuroscience Letters, 1991Co-Authors: Rebecca A. Sheller, Martis L Ballinger, George D. BittnerAbstract:Abstract Glial nuclei have been reported to be incorporated into the Axoplasm of surviving distal stumps (anucleate axons) weeks to months after lesioning abdominal motor axons in rock lobsters. We have not observed this phenomenon in crayfish medial giant axons (MGAs) which also survive for weeks to months after lesioning. Glial nuclei were not observed within MGAs perfused with a physiological intracellular saline. However, incorporation of glial nuclei was observed after MGAs were perfused with intracellular salines containing Fast green. From these and previously published data, we confirm that glial incorporation into Axoplasm can occur, but we suggest that it is not a common mechanism used by crustaceans to provide for long-term survival of anucleate axons.
Rebecca A. Sheller - One of the best experts on this subject based on the ideXlab platform.
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heat shock proteins in Axoplasm high constitutive levels and transfer of inducible isoforms from glia
The Journal of Comparative Neurology, 1998Co-Authors: Rebecca A. Sheller, Mark E. Smyers, Robert M Grossfeld, Martis L Ballinger, George D. BittnerAbstract:To characterize heat-shock proteins (HSPs) of the 70-kDa family in the crayfish medial giant axon (MGA), we analyzed Axoplasmic proteins separately from proteins of the glial sheath. Several different molecular weight isoforms of constitutive HSP 70s that were detected on immunoblots were approximately 1-3% of the total protein in the Axoplasm of MGAs. To investigate inducible HSPs, MGAs were heat shocked in vitro or in vivo, then the axon was bathed in radiolabeled amino acid for 4 hours. After either heat-shock treatment, protein synthesis in the glial sheath was decreased compared with that of control axons, and newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa appeared in both the Axoplasm and the sheath. Because these radiolabeled proteins were present in MGAs only after heat-shock treatments, we interpreted the newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa to be inducible HSPs. Furthermore, the 72-kDa radiolabeled band in heat-shocked Axoplasm and glial sheath samples comigrated with a band possessing HSP 70 immunoreactivity. The amount of heat-induced proteins in Axoplasm samples was greater after a 2-hour heat shock than after a 1-hour heat shock. These data indicate that MGA Axoplasm contains relatively high levels of constitutive HSP 70s and that, after heat shock, MGA Axoplasm obtains inducible HSPs of 72 kDa, 84 kDa, and 87 kDa from the glial sheath. These constitutive and inducible HSPs may help MGAs maintain essential structures and functions following acute heat shock.
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Glia-to-axon communication: enrichment of glial proteins transferred to the squid giant axon.
Journal of neuroscience research, 1995Co-Authors: Rebecca A. Sheller, Michael Tytell, Mark E. Smyers, George D. BittnerAbstract:The transfer of newly synthesized proteins from the glial sheath into the axon is a well-documented process for the squid giant axon. In this study, we used a novel approach to separate the transferred glial proteins (TGPs) from the endogenous Axoplasmic proteins of the squid giant axon. Axoplasm, containing radiolabelled TGPs, was extruded as a cylinder and immersed in an intracellular buffer. After 1–30 min, the TGPs were enriched in the intracellular buffer, because they were eluted from the Axoplasm into the intracellular buffer much faster than the endogenous Axoplasmic proteins. Most of the TGPs enriched in the intracellular buffer did not pellet when centrifuged at 24,000 g for 20 min and were susceptible to protease digestion without the addition of Triton X-100. Additionally, transmission electron microscopic autoradiography of intact axons, containing radiolabelled TGPs, suggested that most TGPs were not associated with vesicular organelles within the axon. We conclude that most of the TGPs are not contained within vesicles in the Axoplasm of the squid giant axon, as would be expected if the mechanism of glia-to-axon transfer were conventional exocytosisendocytosis or microphagocytosis. © 1995 Wiley-Liss, Inc.
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long term survival of severed crayfish giant axons is not associated with an incorporation of glial nuclei into Axoplasm
Neuroscience Letters, 1991Co-Authors: Rebecca A. Sheller, Martis L Ballinger, George D. BittnerAbstract:Abstract Glial nuclei have been reported to be incorporated into the Axoplasm of surviving distal stumps (anucleate axons) weeks to months after lesioning abdominal motor axons in rock lobsters. We have not observed this phenomenon in crayfish medial giant axons (MGAs) which also survive for weeks to months after lesioning. Glial nuclei were not observed within MGAs perfused with a physiological intracellular saline. However, incorporation of glial nuclei was observed after MGAs were perfused with intracellular salines containing Fast green. From these and previously published data, we confirm that glial incorporation into Axoplasm can occur, but we suggest that it is not a common mechanism used by crustaceans to provide for long-term survival of anucleate axons.
Mike Fainzilber - One of the best experts on this subject based on the ideXlab platform.
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Axonal Transport Proteomics Reveals Mobilization of Translation Machinery to the Lesion Site in Injured Sciatic Nerve*□S
2016Co-Authors: Mike FainzilberAbstract:Investigations of the molecular mechanisms underlying responses to nerve injury have highlighted the importance of axonal transport systems. To obtain a comprehensive view of the protein ensembles associated with axonal transport in injured axons, we analyzed the protein com-positions of Axoplasm concentrated at ligatures following crush injury of rat sciatic nerve. LC-MS/MS analyses of iTRAQ-labeled peptides from Axoplasm distal and proxi-mal to the ligation sites revealed protein ensembles trans-ported in both anterograde and retrograde directions. Variability of replicates did not allow straightforward as-signment of proteins to functional transport categories; hence, we performed principal component analysis and factor analysis with subsequent clustering to determine the most prominent injury-related transported proteins
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Axoplasm isolation from rat sciatic nerve.
Journal of visualized experiments : JoVE, 2010Co-Authors: Ida Rishal, Meir Rozenbaum, Mike FainzilberAbstract:Isolation of pure axonal cytoplasm (Axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we demonstrate and describe a protocol for Axoplasm isolation from adult rat sciatic nerve based on the following steps: (1) dissection of nerve fascicles and separation of connective tissue; (2) incubation of short segments of nerve fascicles in hypotonic medium to release myelin and lyse non-axonal structures; and (3) extraction of the remaining axon-enriched material. Proteomic and biochemical characterization of this preparation has confirmed a high degree of enrichment for axonal components.
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Axoplasm isolation from peripheral nerve
Developmental Neurobiology, 2010Co-Authors: Ida Rishal, Meir Rozenbaum, Izhak Michaelevski, Vera Shinder, Katalin F Medzihradszky, Alma L Burlingame, Mike FainzilberAbstract:Localized changes in the composition of axonal cytoplasm (Axoplasm) are critical for many biological processes, including axon guidance, responses to injury, neurite outgrowth, and axon-glia interactions. Biochemical and molecular studies of these mechanisms have been heavily focused on in vitro systems because of the difficulty of obtaining subcellular extracts from mammalian tissues in vivo. As in vitro systems might not replicate the in vivo situation, reliable methods of Axoplasm extraction from whole nerve would be helpful for mechanistic studies on axons. Here we develop and evaluate a new procedure for preparation of Axoplasm from rat peripheral nerve, based on incubation of separated short segements of nerve fascicles in hypotonic medium to separate myelin and lyse nonaxonal structures, followed by extraction of the remaining axon-enriched material. We show that this new procedure reduces serum and glial cell contamination and facilitates proteomic analyses of axonal contents.
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vimentin dependent spatial translocation of an activated map kinase in injured nerve
Neuron, 2005Co-Authors: Eran Perlson, Shlomit Hanz, Rony Seger, Yael Segalruder, Keren Benyaakov, Mike FainzilberAbstract:How are phosphorylated kinases transported over long intracellular distances, such as in the case of axon to cell body signaling after nerve injury? Here, we show that the MAP kinases Erk1 and Erk2 are phosphorylated in sciatic nerve Axoplasm upon nerve injury, concomitantly with the production of soluble forms of the intermediate filament vimentin by local translation and calpain cleavage in Axoplasm. Vimentin binds phosphorylated Erks (pErk), thus linking pErk to the dynein retrograde motor via direct binding of vimentin to importin β. Injury-induced Elk1 activation and neuronal regeneration are inhibited or delayed in dorsal root ganglion neurons from vimentin null mice, and in rats treated with a MEK inhibitor or with a peptide that prevents pErk-vimentin binding. Thus, soluble vimentin enables spatial translocation of pErk by importins and dynein in lesioned nerve.
Scott T. Brady - One of the best experts on this subject based on the ideXlab platform.
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Neurobiology of Disease Pathogenic Forms of Tau Inhibit Kinesin-Dependent Axonal Transport through a Mechanism Involving Activation of
2016Co-Authors: Axonal Phosphotransferases, Yuyu Song, Gerardo Morfini, Gustavo Pigino, Nicholas M. Kanaan, Nichole E. Lapointe, Kristina R. Patterson, Athena Andreadis, Scott T. BradyAbstract:Aggregated filamentous forms of hyperphosphorylated tau (a microtubule-associated protein) represent pathological hallmarks of Alzheimer’s disease (AD) and other tauopathies.While axonal transport dysfunction is thought to represent a primary pathogenic factor in AD and other neurodegenerative diseases, the direct molecular link between pathogenic forms of tau and deficits in axonal transport remain unclear. Recently, we demonstrated that filamentous, but not soluble, forms of wild-type tau inhibit anterograde, kinesin-based fast axonal transport (FAT) by activating axonal protein phosphatase 1 (PP1) and glycogen synthase kinase 3 (GSK3), independent of microtubule binding. Here, we demonstrate that amino acids 2–18 of tau, comprising a phosphatase-activating domain (PAD), are necessary and sufficient for activation of this pathway in Axoplasms isolated from squid giant axons. Various pathogenic forms of tau displaying increased exposure of PAD inhibited anterograde FAT in squid Axoplasm. Importantly, immunohistochemical studies using a novel PAD-specific monoclonal antibody in human postmortem tissue indicated that increased PAD exposure represents an early pathogenic event in AD that closely associates in time with AT8 immunoreactivity, an early marker of pathological tau. We propose a model of pathogenesis in which disease-associated changes in tau conformation lead to increased exposure of PAD, activation of PP1
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Cell Motility and the Cytoskeleton 15:210-219 (1990) Nucleotide Specificity for the Bidirectional Transport of Membrane-Bounded Organelles in Isolated Axoplasm
2016Co-Authors: Philip L. Leopold, George S Bloom, Robert Snyder, Scott T. BradyAbstract:Video microscopy of isolated Axoplasm from the squid giant axon permits corre-lated quantitative analyses of membrane-bounded organelle transport both in the intact Axoplasm and along individual microtubules. As a result, the effects of experimental manipulations on both anterograde and retrograde movements of membrane-bounded organelles can be evaluated under nearly physiological con-ditions. Since anterograde and retrograde fast axonal transport are similar but distinct cellular processes, a systematic biochemical analysis is important for a further understanding of the molecular mechanisms for each. In this series of experiments, we employed isolated Axoplasm of the squid to define the nucleoside triphosphate specificity for bidirectional organelle motility in the axon. Perfusion of Axoplasm with 2-20 mM ATP preserved optimal vesicle velocities in both the anterograde and retrograde directions. Organelle velocities decreased to <50 % of optimal values when the Axoplasm was perfused with 10-20 mM UTP, GTP, ITP, or CTP with simultaneous depletion of endogenous ATP with hexokinase. Under the same conditions, TTP and ATP-y-S were unable to support significant levels of transport. None of the NTPs tested had a differential effect on anterograde vs. retrograde movement of vesicles. Surprisingly, several inconsistencies were re-vealed when a comparison was made between these results and nucleoside triphosphate specificities that have been reported for putative organelle motors by using in vitro assays. These data may be used in conjunction with data from well-defined in vitro assays to develop models for the molecular mechanisms of axonal transport. Key words: organelle motors, nucleoside triphosphates, fast axonal transport, video microscop
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fast axonal transport in isolated Axoplasm from the squid giant axon
Methods in Cell Biology, 2016Co-Authors: Yuyu Song, Gerardo Morfini, Scott T. Brady, Minsu KangAbstract:Abstract The giant axon of the squid provides a unique cell biological model for analyzing the biochemistry and cell biology of the axon. These axons may exceed 500 μm in diameter and can be readily dissected. Once the surrounding small axons and connective tissue are removed, the Axoplasm can be extruded as an intact cylinder of isolated cytoplasm. This isolated Axoplasm is morphologically indistinguishable from the intact axon, but without permeability barriers. Fast axonal transport will continue for more than 4 h after extrusion and can be visualized in real time. By perfusing defined concentrations of proteins and/or reagents into the Axoplasm, this preparation represents a powerful model for study of intracellular trafficking and its underlying molecular mechanisms.
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analysis of microtubules in isolated Axoplasm from the squid giant axon
Methods in Cell Biology, 2013Co-Authors: Scott T. BradyAbstract:Biochemical specialization of cellular microtubules has emerged as a primary mechanism in specifying microtubule dynamics and function. However, study of specific subcellular populations of cytoplasmic microtubules has been limited, particularly in the nervous system. The complexity of nervous tissue makes it difficult to distinguish neuronal microtubules from glial microtubules, and axonal microtubules from dendritic and cell body microtubules. The problem is further compounded by the finding that a large fraction of neuronal tubulin is lost during standard preparations of brain tubulin, and this population of stable microtubules is enriched in axons. Here, we consider a unique biological model that provides a unique opportunity to study axonal microtubules both in situ and in vitro: isolated Axoplasm from the squid giant axon. The Axoplasm model represents a powerful system for addressing fundamental questions of microtubule structure and function in the axon.
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pathogenic forms of tau inhibit kinesin dependent axonal transport through a mechanism involving activation of axonal phosphotransferases
The Journal of Neuroscience, 2011Co-Authors: Nicholas M. Kanaan, Yuyu Song, Gerardo Morfini, Gustavo Pigino, Nichole E. Lapointe, Kristina R. Patterson, Athena Andreadis, Scott T. BradyAbstract:Aggregated filamentous forms of hyperphosphorylated tau (a microtubule-associated protein) represent pathological hallmarks of Alzheimer's disease (AD) and other tauopathies. While axonal transport dysfunction is thought to represent a primary pathogenic factor in AD and other neurodegenerative diseases, the direct molecular link between pathogenic forms of tau and deficits in axonal transport remain unclear. Recently, we demonstrated that filamentous, but not soluble, forms of wild-type tau inhibit anterograde, kinesin-based fast axonal transport (FAT) by activating axonal protein phosphatase 1 (PP1) and glycogen synthase kinase 3 (GSK3), independent of microtubule binding. Here, we demonstrate that amino acids 2-18 of tau, comprising a phosphatase-activating domain (PAD), are necessary and sufficient for activation of this pathway in Axoplasms isolated from squid giant axons. Various pathogenic forms of tau displaying increased exposure of PAD inhibited anterograde FAT in squid Axoplasm. Importantly, immunohistochemical studies using a novel PAD-specific monoclonal antibody in human postmortem tissue indicated that increased PAD exposure represents an early pathogenic event in AD that closely associates in time with AT8 immunoreactivity, an early marker of pathological tau. We propose a model of pathogenesis in which disease-associated changes in tau conformation lead to increased exposure of PAD, activation of PP1-GSK3, and inhibition of FAT. Results from these studies reveal a novel role for tau in modulating axonal phosphotransferases and provide a molecular basis for a toxic gain-of-function associated with pathogenic forms of tau.
Gerardo Morfini - One of the best experts on this subject based on the ideXlab platform.
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Neurobiology of Disease Pathogenic Forms of Tau Inhibit Kinesin-Dependent Axonal Transport through a Mechanism Involving Activation of
2016Co-Authors: Axonal Phosphotransferases, Yuyu Song, Gerardo Morfini, Gustavo Pigino, Nicholas M. Kanaan, Nichole E. Lapointe, Kristina R. Patterson, Athena Andreadis, Scott T. BradyAbstract:Aggregated filamentous forms of hyperphosphorylated tau (a microtubule-associated protein) represent pathological hallmarks of Alzheimer’s disease (AD) and other tauopathies.While axonal transport dysfunction is thought to represent a primary pathogenic factor in AD and other neurodegenerative diseases, the direct molecular link between pathogenic forms of tau and deficits in axonal transport remain unclear. Recently, we demonstrated that filamentous, but not soluble, forms of wild-type tau inhibit anterograde, kinesin-based fast axonal transport (FAT) by activating axonal protein phosphatase 1 (PP1) and glycogen synthase kinase 3 (GSK3), independent of microtubule binding. Here, we demonstrate that amino acids 2–18 of tau, comprising a phosphatase-activating domain (PAD), are necessary and sufficient for activation of this pathway in Axoplasms isolated from squid giant axons. Various pathogenic forms of tau displaying increased exposure of PAD inhibited anterograde FAT in squid Axoplasm. Importantly, immunohistochemical studies using a novel PAD-specific monoclonal antibody in human postmortem tissue indicated that increased PAD exposure represents an early pathogenic event in AD that closely associates in time with AT8 immunoreactivity, an early marker of pathological tau. We propose a model of pathogenesis in which disease-associated changes in tau conformation lead to increased exposure of PAD, activation of PP1
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Inhibition of Fast Axonal Transport by Pathogenic SOD1 Involves Activation of p38 MAP Kinase
2016Co-Authors: Gerardo Morfini, Yuyu Song, Daryl A. Bosco, Hannah Brown, Rodolfo Gatto, Agnieszka Kaminska, Linda Molla, Lisa Baker, Natalia M. Marangoni, Sarah BerthAbstract:Dying-back degeneration of motor neuron axons represents an established feature of familial amyotrophic lateral sclerosis (FALS) associated with superoxide dismutase 1 (SOD1) mutations, but axon-autonomous effects of pathogenic SOD1 remained undefined. Characteristics of motor neurons affected in FALS include abnormal kinase activation, aberrant neurofilament phosphorylation, and fast axonal transport (FAT) deficits, but functional relationships among these pathogenic events were unclear. Experiments in isolated squid Axoplasm reveal that FALS-related SOD1 mutant polypeptides inhibit FAT through a mechanism involving a p38 mitogen activated protein kinase pathway. Mutant SOD1 activated neuronal p38 in mouse spinal cord, neuroblastoma cells and squid Axoplasm. Active p38 MAP kinase phosphorylated kinesin-1, and this phosphorylation event inhibited kinesin-1. Finally, vesicle motility assays revealed previously unrecognized, isoform-specific effects of p38 on FAT. Axon-autonomous activation of the p38 pathwa
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fast axonal transport in isolated Axoplasm from the squid giant axon
Methods in Cell Biology, 2016Co-Authors: Yuyu Song, Gerardo Morfini, Scott T. Brady, Minsu KangAbstract:Abstract The giant axon of the squid provides a unique cell biological model for analyzing the biochemistry and cell biology of the axon. These axons may exceed 500 μm in diameter and can be readily dissected. Once the surrounding small axons and connective tissue are removed, the Axoplasm can be extruded as an intact cylinder of isolated cytoplasm. This isolated Axoplasm is morphologically indistinguishable from the intact axon, but without permeability barriers. Fast axonal transport will continue for more than 4 h after extrusion and can be visualized in real time. By perfusing defined concentrations of proteins and/or reagents into the Axoplasm, this preparation represents a powerful model for study of intracellular trafficking and its underlying molecular mechanisms.
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inhibition of fast axonal transport by pathogenic sod1 involves activation of p38 map kinase
PLOS ONE, 2013Co-Authors: Yuyu Song, Gerardo Morfini, Daryl A. Bosco, Rodolfo Gatto, Agnieszka Kaminska, Hannah E BrownAbstract:Dying-back degeneration of motor neuron axons represents an established feature of familial amyotrophic lateral sclerosis (FALS) associated with superoxide dismutase 1 (SOD1) mutations, but axon-autonomous effects of pathogenic SOD1 remained undefined. Characteristics of motor neurons affected in FALS include abnormal kinase activation, aberrant neurofilament phosphorylation, and fast axonal transport (FAT) deficits, but functional relationships among these pathogenic events were unclear. Experiments in isolated squid Axoplasm reveal that FALS-related SOD1 mutant polypeptides inhibit FAT through a mechanism involving a p38 mitogen activated protein kinase pathway. Mutant SOD1 activated neuronal p38 in mouse spinal cord, neuroblastoma cells and squid Axoplasm. Active p38 MAP kinase phosphorylated kinesin-1, and this phosphorylation event inhibited kinesin-1. Finally, vesicle motility assays revealed previously unrecognized, isoform-specific effects of p38 on FAT. Axon-autonomous activation of the p38 pathway represents a novel gain of toxic function for FALS-linked SOD1 proteins consistent with the dying-back pattern of neurodegeneration characteristic of ALS.
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FALS-linked mutant SOD1 proteins selectively inhibit anterograde, conventional kinesin-dependent FAT.
2013Co-Authors: Gerardo Morfini, Yuyu Song, Daryl A. Bosco, Hannah Brown, Rodolfo Gatto, Agnieszka Kaminska, Linda Molla, Lisa Baker, Natalia M. Marangoni, Sarah BerthAbstract:(a–d) Vesicle motility assays in isolated squid Axoplasm. Individual velocity (µm/sec) rate measurements (arrowheads) are plotted as a function of time (minutes). Dark arrowheads and lines represent anterograde, conventional kinesin-dependent rates. Grey arrows and lines represent retrograde, cytoplasmic dynein-dependent rates. Perfusion of WT-SOD1 protein (a) in Axoplasm shows no effect on either direction. In contrast, perfusion of G93A-SOD1 specifically inhibits anterograde, but not retrograde FAT (b). Similar results were obtained after perfusion of SOD1-G85R (c) and SOD1-H46R (d). n: number of experiments. (e) 30 to 50 minutes after perfusion, anterograde FAT rates were significantly lower in Axoplasms perfused with mSOD1, than in Axoplasms perfused with either WT-SOD1 (#: p≤0.01) or control buffer (not shown). All pathogenic SOD1 proteins tested had no effect on retrograde FAT. (f) Kinesin-1 (KHC IPP) was immunoprecipitated from spinal cord lysates of transgenic mice expressing either WT-SOD1 (WT) or G93A-SOD1 (G93A). An aliquot of lysate (Input) was included as positive control. Immunoblots using anti-kinesin-1 antibody (KHC) demonstrated effective immunoprecipitation from lysates, regardless of genotype. Specificity was confirmed by the absence of kinesin-1 on control immunoprecipitates with non-immune mouse IgG (Ctrl IPP). Immunoblot with an anti-SOD1 monoclonal antibody (D3H5) failed to detect SOD1 in kinesin-1 immunoprecipitates, suggesting that neither WT SOD1 nor G93A-SOD1 interact directly with kinesin-1.